On-Site Inactivation for Disinfection of Antibiotic-Resistant Bacteria in Hospital Effluent by UV and UV-LED

The problem of antimicrobial resistance (AMR) is not limited to the medical field but is also becoming prevalent on a global scale in the environmental field. Environmental water pollution caused by the discharge of wastewater into aquatic environments has caused concern in the context of the sustainable development of modern society. However, there have been few studies focused on the treatment of hospital wastewater, and the potential consequences of this remain unknown. This study evaluated the efficacy of the inactivation of antimicrobial-resistant bacteria (AMRB) and antimicrobial resistance genes (AMRGs) in model wastewater treatment plant (WWTP) wastewater and hospital effluent based on direct ultraviolet (UV) light irradiation provided by a conventional mercury lamp with a peak wavelength of 254 nm and an ultraviolet light-emitting diode (UV-LED) with a peak emission of 280 nm under test conditions in which the irradiance of both was adjusted to the same intensity. The overall results indicated that both UV- and UV-LED-mediated disinfection effectively inactivated the AMRB in both wastewater types (>99.9% after 1–3 min of UV and 3 min of UV-LED treatment). Additionally, AMRGs were also removed (0.2–1.4 log10 for UV 254 nm and 0.1–1.3 log10 for UV 280 nm), and notably, there was no statistically significant decrease (p < 0.05) in the AMRGs between the UV and UV-LED treatments. The results of this study highlight the importance of utilizing a local inactivation treatment directly for wastewater generated by a hospital prior to its flow into a WWTP as sewage. Although additional disinfection treatment at the WWTP is likely necessary to remove the entire quantity of AMRB and AMRGs, the present study contributes to a significant reduction in the loads of WWTP and urgent prevention of the spread of infectious diseases, thus alleviating the potential threat to the environment and human health risks associated with AMR problems.


Introduction
The emergence and spread of antimicrobial-resistant bacteria (AMRB) is becoming an increasingly worldwide problem [1][2][3].The importance of considering not only the environmental health risks and effects of AMRB that are transmitted via humans and animals but also the indirect risks posed to the environment has gained increasing focus [4,5].The O'Neill Commission that was commissioned by the UK government estimates that if effective action is not taken to address the spread of AMRB, annual global deaths will rise from 0.7 million in 2014 to 10 million by 2050, which is more deaths than are caused by cancer, and the economic loss to global GDP is estimated to be $100 trillion [6].These projections are currently becoming a real threat, as recent reports have indicated that the number of AMRB-related deaths has almost doubled to 1.27 million as of 2019 [7].In Japan, an action plan focused on antimicrobial resistance (AMR) has been implemented since 2016 to reduce AMRB prevalence [8,9], and a detailed assessment of the current situation and revision of the action plan to promote further action is underway.
Effluents from hospitals and other healthcare facilities contain a wide variety of microorganisms originating from patients [10][11][12].Antimicrobial use in Japan is approximately 15.8 defined daily doses (DDD) per 1000 adults [13], and this tends to be lower than that of the rest of the world (an average of approximately 20 DDD in Europe and reportedly over 60 DDD in India [14]).In contrast, a worldwide survey of clinical practice reported that drug resistance rates for microorganisms exhibit a typical antimicrobial resistance trend, including penicillin-resistant Streptococcus pneumoniae (PRSP) at 48%, methicillinresistant Staphylococcus aureus (MRSA) at 51%, carbapenem-resistant Pseudomonas aeruginosa (CRPA) at 17%, and third-generation cephalosporin-resistant Escherichia coli (CREC) at 18% [4,[15][16][17].Among these, Japan possesses one of the highest rates of resistance in the world to PRSP and MRSA, whereas CRPA and CREC are also at high levels [18].Therefore, understanding the actual AMRB in medical wastewater, assessing the risk to the environment, and developing technologies that can reduce or eliminate such risks can contribute not only to protecting human health but also to reducing nosocomial infections and improving the quality of healthcare [19][20][21].Additionally, the AMRB issue is also important in regard to ensuring the safety of the water environment and the protection of watersheds on a large scale, and it is also considered an important issue in the context of balancing sustainable human prosperity and global environmental protection [2,22,23].
Several methods are known to inactivate microorganisms in wastewater, including chlorination, Fenton, ozone, and photocatalytic oxidation [24][25][26].All of these methods are known to be effective in inactivating microorganisms and have been studied for adaptation to hospital wastewater treatment methods [21,27].However, they require the addition of chemicals for treatment, and the residual chemicals after treatment are often an issue [28,29].On the other hand, disinfection with ultraviolet light (UV) is effective for inactivating microorganisms without the addition of chemicals that are resistant to chlorine disinfection such as Cryptosporidium and other microorganisms [30,31].UV disinfection primarily uses UV lamps that emit low-wavelength (254 nm) UV light that is considered to be highly effective in inactivating microorganisms.However, UV lamps use mercury discharge to generate ultraviolet light, and therefore, the environmental impact of mercury use cannot be ignored.Additionally, the voltage required to drive the lamps is high, maintenance costs are high due to the short lamp life, and there is not much flexibility in the design of the treatment equipment [32,33].Recently, ultraviolet light-emitting diodes (UV-LEDs) have undergone rapid technological development in recent years and possess many excellent advantages such as mercury-free functionality, low voltage requirements, and a 10-fold longer life span than UV lamps.Currently, their future use as a replacement for UV lamps is becoming a reality [32,34,35].
The low-wavelength UV light that can be emitted by UV-LEDs currently on the market is at 255, 265, 280, 300, and 365 nm.However, it has been reported that UV irradiation doses at 255 nm and 265 nm are extremely low compared to UV lamps using existing technology, and problems remain due to equipment limitations when considering implementation and application in a stand-alone disinfection process [36][37][38].Additionally, ultraviolet rays above 300 nm are the same as long-wave ultraviolet rays (>290 nm) [39] that are close to the visible light contained in sunlight reaching the surface of the Earth and are not suitable for use as an inactivation treatment for large volumes of water such as wastewater in terms of disinfection effectiveness [40,41].Therefore, when considering alternatives to 254 nm UV lamps that are known to be effective in regard to inactivating microorganisms, evaluations have primarily focused on a new UV wavelength of 280 nm that is believed to be capable of providing high-power UV irradiation at lower wavelengths [34,42].To date, research has led to the evaluation of inactivation effects and the development of methods for a wide variety of environmental contaminants such as dyes, pesticides, and pharmaceuticals [32,43,44] and also for common indicator microbes such as E. coli and Enterococci [45][46][47], viruses [30], fungi [38], and COVID-19 [48,49].However, only a limited number of studies have attempted to evaluate the inactivation of AMRB in wastewater using UV-LED.
There is a worldwide lack of knowledge on the study of hospital wastewater, and few reports on the actual status of environmental pollutants and their impact on the environment, as well as on treatment [50][51][52].The treatment of hospital wastewater at the source with high concentrations is more effective in reducing the impact of environmental pollutants on the environment than treating large volumes of diluted wastewater at WWTPs, as is the case with many other types of wastewater [53][54][55].Therefore, evaluating the effectiveness and efficiency of disinfection treatments using UV lamps and UV-LED to inactivate AMRB in hospital wastewater is expected to provide a more comprehensive understanding of the emerging water pollution problem caused by AMRB and yield valuable insights for predicting and assessing the environmental risks associated with the release of AMRB into the environment.
In this study, to evaluate the effectiveness of UV lamps and UV-LED in the context of disinfection treatment of pharmaceutical bacteria in wastewater, treatment tests were first conducted using wastewater obtained from a wastewater treatment plant (WWTP) and wastewater obtained from a hospital as wastewater from a medical facility, and the inactivation effect was evaluated.Next, based on the results obtained, the effectiveness of UV-LED as a new disinfection treatment method for environmental risk reduction by comparing the effectiveness of AMRB inactivation by UV and UV-LED and by assessing the effectiveness of using UV-LED on medical wastewater was evaluated.

Sampling
Wastewater samples were collected from a WWTP and a hospital located in an urban area of Japan as described previously [56].The WWTP treats municipal sewage generated by a population of 420,000 individuals.The WWTP influent was first treated with conventional activated sludge and discharged as the WWTP secondary effluent.The WWTP secondary effluent was treated with chlorine (1.2 mg NaClO/L for 15 min) for disinfection and then discharged into the river as WWTP effluent.Hospital effluent was collected directly from a hospital with 480 beds and an average of 1200 patients per day.Hospital effluent is discharged into municipal sewage, merged with domestic and industrial wastewater, and eventually introduced into the WWTP influent.Samples were collected in December 2022 on the days when the influence of precipitation was low and when the recorded rainfall was >1 mm for the preceding two days [57].Basic water quality parameters of WWTP wastewater and hospital effluent were pH 7.5 and 8.4, 19 mg/L and 51 mg/L for chemical oxygen demand (COD), 5.2 mg/L and 74 mg/L for biochemical oxygen demand (BOD), 12 mg/L and 204 mg/L for suspended solids (SS), 2.4 mg/L and 30 mg/L for NH 4 -N, 1.0 mg/L and 2.3 mg/L for PO 4 3− -P, 167 CFU/mL and 2600 CFU/mL for E. coli, and 617 CFU/mL and 108,000 CFU/mL for coliform bacteria, respectively.A stainless-steel pail sampler was used to collect the wastewater samples, which were then placed into separate sterilized glass bottles.Sodium thiosulfate (0.5 g/L) was immediately added to each bottle to quench the residual chlorine [58,59].All samples were immediately transported to the laboratory in a cooler box (within 2 h), stored at 4 • C in the dark, and processed within 12 h.

Disinfection of Wastewater by UV and UV-LED
Inactivation testing of AMRB and antimicrobial-susceptible bacteria (AMSB) using UV and UV-LED was based on previous reports of inactivation of microorganisms by the UV-LED system [45,60,61] and also on various guidelines for the light attenuation of environmental contaminants [62][63][64].The hospital effluent was dispensed into a sterilized glass Petri dish with a diameter of 120 mm and an effective volume of 220 mL and maintained in the dark at 20 • C, as this is the annual mean water temperature of WWTPs in Japan [65].
UV irradiation was performed using a low-pressure mercury lamp (3UV-38; Funakoshi Co. Ltd., Tokyo, Japan) at a peak wavelength of 254 nm.UV-LED irradiation was supplied by a UV-LED system (PearlBeam, NIKKISO Co., Ltd., Tokyo, Japan) with peak emission at 280 nm.The UV irradiation intensity (mW/cm 2 ) was measured within the UV region (240-480 nm) using a UV radiometer (UVPadE; Opsytec Dr. Gröbel GmbH, Ettlingen, Germany).UV irradiation was performed by irradiating the dish with UV light from a UV or UV-LED source that was attached to the top of the dishes [34,47].The UV and UV-LED irradiation systems are presented in Figure S1.UV and UV-LED irradiation intensities were fixed by adjusting the distance between the irradiator and the dish to ensure that the same irradiation intensity (0.3 mW/cm 2 for UV and 0.3 mW/cm 2 for UV-LED, which is approximately the maximum intensity value of UV-LED irradiation intensity) was used in the inactivation experiments.Samples were collected after 0, 0.2, 0.3, 0.5, 1, and 3 minutes of exposure.
In a similar previous study, no significant differences (p < 0.05) were observed for proliferation or attenuation for both AMRB and AMSB at 7 h at 20 • C under dark conditions [66].As the inactivation treatment time used in this study was much shorter, the present investigation directly expressed the inactivation effect of microorganisms based on the results obtained by UV irradiation.The duration of inactivation was determined based on the average treatment conditions (3.6 ± 1.8 min and 277 ± 164 J/m 2 as UV fluences) in WWTPs that use UV disinfection in Japan [65] and on the values reported in previous studies [38,45,60].In this study, the UV fluence (mJ/cm 2 ) was calculated by multiplying the UV irradiation intensity (mW/cm 2 ) by the inactivation treatment time (min) and the transmittance of UV light in the water samples [67,68].Finally, the solutions were analyzed using chromogenic agar methods as described in Section 2.3.

Microbial Analysis
Six representative classes of AMRB have been assigned a global priority by the WHO [69,70] and addressed in the clinical area [5,71,72] and five classes of antimicrobialsusceptible bacteria (AMSB) used as the microbial species comprising each ARB that was targeted were also investigated as reported previously [66,73].The lists and abbreviations of the target AMRB and AMSB are presented in Table 1.The prevalence of each type of AMRB and AMSB in the wastewater samples was determined by screening individual microbes grown on chromogenic agar formulations.ChromID CARBA was used for the detection of CRE, chromID ESBL was used for the detection of ESBL-E, chromID MRSA was used for the detection of MRSA, ChromID VRE New was used for the detection of VRE (bioMérieux S.A., Marcy-l'Étoile, France), CHRO-Magar MDRA was used for the detection of MDRA, and CHROMagar MDRP was used for the detection of MDRP (Kanto Chemical Co., Inc., Tokyo, Japan).Similarly, the amount of AMSB was estimated by screening for individual microbes grown on different chromogenic agar formulations devoid of antimicrobials.CHROMagar Acinetobacter was used for the detection of Acinetobacter, CHROMagar Pseudomonas was used for the detection of Pseudomonas aeruginosa (P.aeruginosa) (Kanto Chemical Co., Inc., Tokyo, Japan), chromID S. aureus Elite was used for the detection of Staphylococcus aureus (S. aureus), chromID CPS Elite was used for the detection of Enterococcus (bioMérieux S.A., Marcy-l'Étoile, France), and XM-G agar was used for the detection of E. coli (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan).AMRB and AMSB were detected following the protocols provided by the manufacturers of the growth media using methods [74] that were described previously [75][76][77][78][79][80].Ultrapure Milli-Q water (18.2MΩ•cm; MilliporeSigma, Watford, UK) with a pH adjusted to 7.0 and a 10 M sterilized phosphate buffer were used for dilution.
An aliquot (1 mL) of the water sample was poured onto each agar plate and quickly spread over the surface.The wastewater was aspirated several times with a pipette prior to collection and mixed well before the treated water sample was collected for measurement.Each plate was then covered with a cover plate and incubated at 42 ± 1 • C for 24 h for CRE and ESBL-E and at 37 ± 1 • C for 24 h for the other bacteria in the dark.The bacterial species were differentiated based on the color and morphology of the colony in accordance with the manufacturer's specifications and as described previously [74,81,82].The experiments were conducted in triplicate for each case.Colonies were counted, and the number of bacteria that were recovered was expressed as colony-forming units per milliliter (CFU/mL).This was then converted into mean yearly values.If the mean CFU was a whole number, the values were expressed as the nearest integer after application of the rounding-off rule and were counted as N.D. (not detected) if the values were <1.The relative reproducibility values (n = 3) for the AMRB (CRE, ESBL, MDRA, MDRP, MRSA, and VRE) and AMSB (Acinetobacter, Enterococcus, E. coli, P. Aeruginosa, and S. aureus) were 13%, 9%, 10%, 19%, 11%, and 13% and 14%, 12%, 16%, 9%, and 19%, respectively.

Quantitative PCR (qPCR) Analysis
Quantification of β-lactam resistance genes (bla IMP , bla TEM , and bla CTX-M ) that are frequently encountered in clinical sites for CRE (bla IMP ) and ESBL (bla TEM and bla CTX-M ) as representative ARGs was performed using Quantitative PCR (qPCR) analysis as described previously [11,56,83].Briefly, 50 mL aliquots of wastewater samples were concentrated using a membrane filter (0.45-µm pore size, Millipore Sigma, Burlington, MA, USA), and genomic DNA was extracted using a DNeasy ® Blood & Tissue Kit (QIAGEN, Hilden, Germany) [84][85][86].Sample pretreatment was conducted using TB Green Premix Ex Taq II (Tli RNaseH Plus; Takara Bio Inc., Shiga, Japan) in 20 µL reaction mixtures containing 3 µL of the DNA template and 0.4 µM of each primer (Table S1) [87][88][89][90].qPCR was performed using a LightCycler ® 480 System II instrument and its respective software (Roche Diagnostics, Basel, Switzerland).Reaction conditions were as follows: initial denaturation at 95 • C (30 s) was followed by annealing for 45 cycles at 95 • C (3 s each) and elongation at appropriate temperatures (57-62 • C) for 30 s.The copy numbers of the bacterial 16S rRNA genes were also determined from the same extracts to normalize values across samples and compare the bacterial abundance between each wastewater sample [91][92][93].The primers and standard curves that were used to validate the amplification efficiency and linearity (r 2 ) of each gene are summarized in Table S1.The concentrations of the plasmid stock solutions extracted to make the standard curve were 4.5 × 10 10 , 3.2 × 10 10 , 3.1 × 10 10 , and 3.2 × 10 10 copies/µL for bla IMP , bla TEM , bla CTX-M , and 16S rRNA, respectively.A standard curve was prepared for the plasmid stock solution by making a 10-fold staircase dilution and using it for qPCR.Sample concentrations were within the range of the standard curve.Analyses were conducted in duplicate, and the mean and standard error were calculated.

Bacterial Community Structure Analysis
Bacterial community structure analysis was performed using MiSeq platform analysis as described previously [94][95][96].Genomic DNA was extracted from the water samples using an Extrap Soil DNA Kit Plus v.2 (Nippon Steel Eco-Tech Corporation, Tokyo, Japan), and DNA concentrations and purifications were assessed using a Qubit ® 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit ® dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) [97,98].The V1-V2 region of the bacterial 16S ribosomal RNA (rRNA) gene was used to characterize the bacterial communities [99,100], and the universal bacterial primers 27F/338R were used for PCR amplification [101,102].PCR was performed using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA).Finally, the amplified genes were sequenced on a MiSeq platform (Illumina Inc., San Diego, CA, USA) according to the manufacturer's instructions and previous reports [103][104][105].Sequence data were pre-processed and analyzed using the Flora Genesis software v.20161108 (Repertoire Genesis Inc., Osaka, Japan).Operational taxonomic units (OTUs) were selected using the open-reference method at a 97% identity level and annotated from the prefiltered Greengenes Database v.13.8 by the UCLUST algorithm [106,107], and taxonomy was assigned using the Ribosomal Database Project classifier at a confidence threshold of 0.80 [108,109].

Statistical Analysis
The data for the tested traits were analyzed using Microsoft Excel software (Office 2019) and are presented as mean values with their individual standard deviation values.A paired t-test was conducted to evaluate the difference in inactivation rates between water samples with a statistical significance of p < 0.05 [110,111].

Disinfection of the WWTP Wastewater and Hospital Effluent by UV and UV-LED Treatment
The current situation regarding the occurrence of AMRB in the WWTP (influent, secondary effluent, and effluent) and hospital effluent is presented in Table 2.All AMRB targeted in this study were detected in all wastewater samples.The concentrations of AMRB and AMSB ranged from 35 CFU/mL to 963 CFU/mL and 50 CFU/mL to 20,000 CFU/mL in the WWTP influent, from N.D. to 17 CFU/mL and N.D. to 664 CFU/mL in the secondary effluent, from N.D. to 17 CFU/mL and 1 CFU/mL to 140 CFU/mL in the WWTP effluent, and from 31 to 215 CFU/mL and 63 CFU/mL to 13,000 CFU/mL in the hospital effluent, respectively.These results revealed that AMRB was widely present in the wastewater and could be mostly removed during the wastewater treatment process at the WWTP, although some of them were discharged into the river as effluent.These concentrations are typically within a range similar to those described in previous reports on antimicrobials in hospital wastewater in Japan [66].
The time-dependent inactivation profiles of AMRB and AMSB in WWTP wastewater during UV and UV-LED treatments are presented in Figure 1.Although the inactivation time differed among the bacteria, all targeted AMRB and AMSB present in WWTP wastewater were inactivated by both UV and UV-LED.Inactivation of AMRB and AMSB by UV and UV-LED followed pseudo-first-order kinetics as previously reported for the UV disinfection of multiple bacteria and viruses [68,[112][113][114].The majority of the AMRB (CRE, ESBL-E, MDRA, MDRP, and VRE) and AMSB (Acinetobacter, Enterococccus, E. coli, and P. aeruginosa) with the exception of MRSA and S. aureus in the WWTP wastewater were rapidly inactivated at the level of >90% after 0.3 min, and this level reached >99% after 0.5 min UV treatment.In the case of the UV-LED treatment, the time required for inactivation tended to be longer than that for the UV treatment; however, the same inactivation effect on microorganisms was observed with the UV treatment.No significant differences (p < 0.05) were observed in the effects of UV irradiation on the AMRB and AMSB.The inactivation of MRSA and S. aureus progressed more slowly than did that of other microorganisms in both UV and UV-LED treatments and required 1 min for UV and greater than 3 min for UV-LED to achieve >90% inactivation.The rigidity of the cell wall structure of MRSA and S. aureus may be the likely reason for their resistance to UV inactivation, as the cell walls of MRSA and S. aureus are stronger than those of other bacteria.This makes them resistant to multiple environmental conditions [115,116] and chlorine disinfection [117,118].Therefore, it will be a challenge to try to analyze the models and mechanisms of the inactivation reactions for each microorganism in the future.These results demonstrate the effectiveness of UV inactivation of AMRB and AMSB in both WWTP and hospital wastewater.The mechanism of inactivation of microorganisms by UV is considered to be mainly due to inhibition of gene transcription by dimerization of nucleobases in microbial DNA [121][122][123].On the other hand, the inactivation mechanisms need to be elucidated at the molecular level due to the various structural and biological differences among microorganisms.Further developments need to be addressed for the inactivation mechanism of AMRB and AMSB with different wavelengths of ultraviolet light including UV-LEDs in the near future.The inactivation profiles of AMRB and AMSB in hospital effluent during UV and UV-LED treatment are presented in Figure 2. When UV or UV-LED disinfection was applied to the hospital effluent, the time required for inactivation tended to increase; however, the trend of inactivation for the various suborgins was similar to the results obtained with the wastewater treatment plant effluent.Greater than 90% of AMRB and AMSB with the exception of MRSA and S. aureus were inactivated after 0.5 min in UV treatment and after 0.5 min in UV-LED treatment, respectively.Conversely, MRSA and S. aureus were gradually inactivated at 54, 66, and 84% for the former and at 49, 60, and 88% for the latter in response to UV treatment and at 44, 29, and 54% for the former and at 0, 54, and 80% for the latter in response to UV-LED treatment.These inactivation levels occurred after 0.5-, 1-, and 3-min treatments and reached the level of >99% after 3 mins of treatment, respectively.Interestingly, the inactivation effect obtained in this study tended to be mainly microorganisms that fit the regression curve, whereas in the case of Enterococcus, the fit to the straight line was less pronounced.These results would be due to the fact that Enterococcus is composed of a complex of many different microorganisms and has different characteristics compared to other microorganisms, which tends to deviate the inactivation system from a simple exponential linear system.This trend was also observed in a similar study on the inactivation of microorganisms in wastewater [119,120].Therefore, it will be a challenge to try to analyze the models and mechanisms of the inactivation reactions for each microorganism in the future.

Inactivation Kinetics of AMRB and AMSB in the WWTP Wastewater and Hospital Effluent by UV and UV-LED Treatment
The distribution of the inactivation rates for AMRB and AMSB in response to UV and UV-LED treatments is summarized in Table 3, and the estimated distribution of the halflives of the antimicrobials is presented in Table S2.The mean inactivation rate constants for AMRB and AMSB in WWTP wastewater were 6.6 ± 2.7 min −1 for UV and 7.2 ± 2.8 min −1 for UV-LED, and in hospital effluent, they were 3.2 ± 1.6 min −1 and 4.0 ± 1.8 min −1 , respectively.Additionally, the mean inactivation rate constants for AMRB and AMSB in hospital effluent were 3.9 ± 2.7 min −1 for UV and 4.8 ± 2.4 min −1 for UV-LED, and in hospital effluent, these values were 3.4 ± 4.3 min −1 and 2.2 ± 0.9 min −1 , respectively.The estimated half-lives typically ranged from <0.1 to 0.9 min.Detailed distributions of the half-lives of AMRB and AMSB are summarized in Table S2.These results demonstrate the effectiveness of UV inactivation of AMRB and AMSB in both WWTP and hospital wastewater.The mechanism of inactivation of microorganisms by UV is considered to be mainly due to inhibition of gene transcription by dimerization of nucleobases in microbial DNA [121][122][123].On the other hand, the inactivation mechanisms need to be elucidated at the molecular level due to the various structural and biological differences among microorganisms.Further developments need to be addressed for the inactivation mechanism of AMRB and AMSB with different wavelengths of ultraviolet light including UV-LEDs in the near future.

Inactivation Kinetics of AMRB and AMSB in the WWTP Wastewater and Hospital Effluent by UV and UV-LED Treatment
The distribution of the inactivation rates for AMRB and AMSB in response to UV and UV-LED treatments is summarized in Table 3, and the estimated distribution of the halflives of the antimicrobials is presented in Table S2.The mean inactivation rate constants for AMRB and AMSB in WWTP wastewater were 6.6 ± 2.7 min −1 for UV and 7.2 ± 2.8 min −1 for UV-LED, and in hospital effluent, they were 3.2 ± 1.6 min −1 and 4.0 ± 1.8 min −1 , respectively.Additionally, the mean inactivation rate constants for AMRB and AMSB in hospital effluent were 3.9 ± 2.7 min −1 for UV and 4.8 ± 2.4 min −1 for UV-LED, and in hospital effluent, these values were 3.4 ± 4.3 min −1 and 2.2 ± 0.9 min −1 , respectively.The estimated half-lives typically ranged from <0.1 to 0.9 min.Detailed distributions of the half-lives of AMRB and AMSB are summarized in Table S2.When comparing the inactivation effects of UV and UV-LED treatments, statistically significant differences (p < 0.05) were observed for MDRP, P. aeruginosa, E. coli, and Enterococccus in WWTP wastewater, and ESBL and P. aeruginosa in hospital effluent, but not for other microorganisms.Table 4 summarizes the estimated fluences required to inactivate 99% of each microorganism based on the inactivation of AMRB and AMSB over time.The results revealed that the fluences required for inactivation of AMRB and AMSB were 16 ± 6 mJ/cm 2 and 14 ± 10 mJ/cm 2 for UV and 37 ± 22 mJ/cm 2 and 30 ± 17 mJ/cm 2 for UV-LED for WWTP wastewater and 10 ± 5 mJ/cm 2 and 8 ± 5 mJ/cm 2 for UV and 31 ± 22 mJ/cm 2 and 23 ± 12 mJ/cm 2 for UV-LED for hospital effluent, respectively.A trend of 1.5-to 2-fold higher fluences required for the inactivation of different microorganisms was observed for UV-LED compared to that for UV.However, statistically significant differences (p < 0.05) were observed between the two for the fluences required for the inactivation of MDRA and P. aeruginosa in WWTP wastewater and MDRP and Enterococcus in hospital effluent, thus suggesting that the characteristic differences in susceptibility attributable to bacterial species between UV and UV-LED light tended to be small.The technological development of diodes that are used to make UV-LEDs has progressed rapidly worldwide, and it is expected that UV irradiation will become possible with a much higher output power [32,45,124].The efficacy and effectiveness of UV-LEDs remain largely unknown, and further detailed studies are required before these sources can be of practical use [30,40,125].The results of this study are of interest in the context of the investigation of low-energy, long-life UV-LEDs as mercury-free alternatives to UV light.In addition, the fluences required for the inactivation of AMRB and AMSB will help us to evaluate the treatment condition for effective inactivation of AMRB and AMSB in wastewater.The present results support the need for further conclusive research that considers experimental, technical, and regional customs, biases, and other unknown factors.The relative abundances of the resistome profiles of each wastewater sample are presented in Figure 4 and Table S3.The distribution of genes corresponding to the different AMRGs in the effluent before treatment was −5.4 ± −6.4 log10 (AMRGs/16S rRNA gene) for blaIMP, 2.8 ± 3.3 log10 (AMRGs/16S rRNA gene) for blaTEM, and 5.8 ± 6.9 log10 (AMRGs/16S rRNA gene) for blaCTX-M genes.These results were similar to those obtained abroad (−5 to −1 log10 [AMRGs/16S rRNA gene]) [134][135][136].AMRGs after various types of ozone treatment for blaIMP, blaTEM, and blaCTX-M in response to UV were −3.9 ± −4.2, −5.7 ± −6.2, and −6.7 ± −7.0 and in response to UV-LED were −4.0 ± −4.5, −5.7 ± −6.5, and −6.6 ± −6.8 for WWTP wastewater, and these values were −5.2 ± −5.4,−2.7 ± −3.6, and −5.3 ± −5.5 and −5.1 ± −5.9, −2.8 ± −3.4, and −5.3 ± −5.7 for hospital effluent, respectively.These results suggest that although AMRGs are removed by UV-based treatments, they remain in the river environment with less significant changes in their morphology [132,137].The DNA of AMRB that did not survive in the water samples was accounted for by viable AMRB as UV and UV-LED processes tended to remove AMRGs with removal rates of 1.1 log 10 for bla IMP , 1.1 log 10 for bla TEM , and 1.4 log 10 for bla CTX-M in UV, while for the UV-LED processes, the removal rate was 1.3 log 10 for bla IMP , 1.2 log 10 for bla TEM , and 1.3 log 10 for bla CTX-M in WWTP wastewater.In hospital effluent, removal rates of AMRGs were 0.5 log 10 for bla IMP , 0.7 log 10 for bla TEM , 0.2 log 10 for bla CTX-M in UV, and 0.3 log 10 for bla IMP , 0.6 log 10 for bla TEM , and 0.1 log 10 for bla CTX-M in UV-LED processes, respectively.Notably, there was no statistically significant decrease (p < 0.05) in AMRGs between the UV and UV-LED treatments, although the DNA of AMRB that did not survive in the water samples was accounted for according to detected genes [126,127].In contrast, none of the AMRGs targeted in this study were completely removed, and some tended to remain after the treatment.Previously reported ozone treatment was effective in treating both viable AMRB and AMRGs as genes, the reason would be related to the strong oxidizing potential of ozone, which is not seen in UV and UV-LED treatment [128][129][130].This will be an important issue when considering the environmental impacts of AMRGs [131][132][133].
Antibiotics 2024, 13, x FOR PEER REVIEW 12 of 21 detected genes [126,127].The present results support the need for further conclusive research that considers experimental, technical, and regional customs, biases, and other unknown factors.
Several researchers have been concerned regarding the persistence of AMRGs in conventional wastewater treatment processes such as biological treatment and chlorine disinfection in wastewater treatment plants (WWTPs) and water environments.[68,[137][138][139][140].The potential effects of AMRGs in an aqueous environment are due to a wide variety of microorganisms in activated sludge in biological treatment reactors at WWTPs [141], and there have been concerns that AMRB could result in a pool of antimicrobial-resistant bacteria through zygotic transmission or transformation [94,142,143].Additionally, the effect of gene transfer and transformation on the potential emergence of new AMRB must be considered [144][145][146].Therefore, detailed and quantitative assessments of environmental risks to ecosystems and human health risks from both AMRB and AMRGs in river environments are considered important issues in the near future [147][148][149][150][151]. One way to overcome these challenges is to introduce the types of advanced water treatment systems that have been examined in this study.Additionally, there have been a growing number of studies in recent years focused on reducing or eliminating the environmental impact of antimicrobials, AMRB, and AMSB in wastewater discharged into public sewers by on-site treatment at hospital facilities before they are aggregated and diluted for discharge into sewers [1,20,117].Recently, in addition to the UV treatments covered in this study, UV chlorine [123,152], ozone [73,153], electrochemical [154,155], peracetic acid [156,157], and membrane [158,159] treatments appear to be effective in regard to decreasing the levels of these new environmental pollutants in wastewater discharged into the water environment.

Bacterial Community Structure Analysis
The bacterial community structures in the WWTP wastewater and hospital effluent before and after UV and UV-LED treatment based on the taxonomic affiliation of the OTUs are summarized in Figure 5.A total of 147,940, 154,719, and 156,556 bacterial 16S rRNA reads were obtained from WWTP wastewater upon commencement of UV and UV-LED treatment, respectively (7222 OTUs in total).From the hospital effluent, 140,437, 125,587, and 121,287 reads were collected from the water samples upon the commencement of treatment and after UV and UV-LED treatments, respectively (2954 OTUs in total).WWTP wastewater contained 42 bacterial phyla, 132 classes, 232 orders, 395 families, and 768 genera.The hospital effluent contained 25 bacterial phyla, 50 classes, 91 orders, 172 families, and 385 genera.Additionally, the major phyla were Proteobacteria (44%), Several researchers have been concerned regarding the persistence of AMRGs in conventional wastewater treatment processes such as biological treatment and chlorine disinfection in wastewater treatment plants (WWTPs) and water environments [68,[137][138][139][140].The potential effects of AMRGs in an aqueous environment are due to a wide variety of microorganisms in activated sludge in biological treatment reactors at WWTPs [141], and there have been concerns that AMRB could result in a pool of antimicrobial-resistant bacteria through zygotic transmission or transformation [94,142,143].Additionally, the effect of gene transfer and transformation on the potential emergence of new AMRB must be considered [144][145][146].Therefore, detailed and quantitative assessments of environmental risks to ecosystems and human health risks from both AMRB and AMRGs in river environments are considered important issues in the near future [147][148][149][150][151].
One way to overcome these challenges is to introduce the types of advanced water treatment systems that have been examined in this study.Additionally, there have been a growing number of studies in recent years focused on reducing or eliminating the environmental impact of antimicrobials, AMRB, and AMSB in wastewater discharged into public sewers by on-site treatment at hospital facilities before they are aggregated and diluted for discharge into sewers [1,20,117].Recently, in addition to the UV treatments covered in this study, UV chlorine [123,152], ozone [73,153], electrochemical [154,155], peracetic acid [156,157], and membrane [158,159] treatments appear to be effective in regard to decreasing the levels of these new environmental pollutants in wastewater discharged into the water environment.

Bacterial Community Structure Analysis
The bacterial community structures in the WWTP wastewater and hospital effluent before and after UV and UV-LED treatment based on the taxonomic affiliation of the OTUs are summarized in Figure 5.A total of 147,940, 154,719, and 156,556 bacterial 16S rRNA reads were obtained from WWTP wastewater upon commencement of UV and UV-LED treatment, respectively (7222 OTUs in total).From the hospital effluent, 140,437, 125,587, and 121,287 reads were collected from the water samples upon the commencement of treatment and after UV and UV-LED treatments, respectively (2954 OTUs in total). in susceptible hosts, and it can survive in aquatic environments [161,162].
Bacterial community structure was not apparently affected during both UV and UV-LED treatment for the WWTP wastewater (UV: Proteobacteria Firmicutes [17%], Actinobacteria [1%], and TM7 [0.1%]).Overall, the present results suggest the importance of introducing advanced wastewater treatment for the removal of AMRB and AMRGs, although some bacteria are not completely removed [163].This appears to be reasonable considering the existence of multiple microorganisms [138,163,164] and AMRGs [165][166][167][168] in wastewater and river water.The results of the present study suggest that comprehensive removal of AMRB, including AMRGs, requires the introduction of advanced wastewater treatment systems when considering multiple microorganisms [138,163,164] and AMRGs [165][166][167][168]. Recent research has provided insights into the environmental risk of both AMRB and AMRGs [169][170][171].The environmental risk of infection by AMRB in water and via the ecosystem and the further development of AMRB in the presence of residual antimicrobials or AMRGs in water are currently being assessed [148,149].Furthermore, the present study provides valuable information for preventing infectious diseases in aquatic environments, including wastewater.To the best of our knowledge, this is the first report demonstrating the inactivation profiles of AMRB and AMRGs in hospital WWTP wastewater contained 42 bacterial phyla, 132 classes, 232 orders, 395 families, and 768 genera.The hospital effluent contained 25 bacterial phyla, 50 classes, 91 orders, 172 families, and 385 genera.Additionally, the major phyla were Proteobacteria (44%), Bacteroidetes (38%), Firmicutes (13%), Fusobacteria (2%), and TM7 (0.8%) in WWTP wastewater and Proteobacteria (58%), Bacteroidetes (27%), Firmicutes (14%), Actinobacteria (1%), and TM7 (0.1%) in hospital effluent.Proteobacteria comprise the majority of the composition of both WWTP wastewater and hospital effluents and are considered to be one of the most diverse and abundant groups of microbes on earth with low pathogenic potential [160].This finding suggests that environmental bacteria comprise the majority of microorganisms when considering hospital wastewater as a whole and that microorganisms such as AMRB that are pathogenic and infectious and require clinical attention comprise only a small proportion [12,23].Conversely, Bacteroides is a biotactic anaerobic gram-negative bacillus that is endemic to the human colon.It is also an opportunistic infectious organism that causes intra-abdominal abscesses and septicemia in susceptible hosts, and it can survive in aquatic environments [161,162].
Bacterial community structure was not apparently affected during both UV and UV-LED treatment for the WWTP wastewater (UV: Proteobacteria Actinobacteria [1%], and TM7 [0.1%]).Overall, the present results suggest the importance of introducing advanced wastewater treatment for the removal of AMRB and AMRGs, although some bacteria are not completely removed [163].This appears to be reasonable considering the existence of multiple microorganisms [138,163,164] and AMRGs [165][166][167][168] in wastewater and river water.The results of the present study suggest that compre-hensive removal of AMRB, including AMRGs, requires the introduction of advanced wastewater treatment systems when considering multiple microorganisms [138,163,164] and AMRGs [165][166][167][168].
Recent research has provided insights into the environmental risk of both AMRB and AMRGs [169][170][171].The environmental risk of infection by AMRB in water and via the ecosystem and the further development of AMRB in the presence of residual antimicrobials or AMRGs in water are currently being assessed [148,149].Furthermore, the present study provides valuable information for preventing infectious diseases in aquatic environments, including wastewater.To the best of our knowledge, this is the first report demonstrating the inactivation profiles of AMRB and AMRGs in hospital wastewater using direct UV and UV-LED treatments.These results will improve the understanding of the environmental pollution associated with AMRB and AMRGs in aquatic environments.The introduction of any advanced wastewater treatment system that can be used in conjunction with UV radiation such as ozonation at medical facilities and WWTPs may provide a one-step forward measure for the reduction of any risks caused by pollution in aquatic environments.

Conclusions
This study investigated the effectiveness of UV and UV-LED treatments for the inactivation of AMRB and AMRGs in model WWTP wastewater and real hospital wastewater.Overall, the results indicate that direct UV and UV-LED treatments inactivated the majority of AMRB and partially removed AMRGs.No significant differences were observed in the effects of UV irradiation on genes that comprise these microorganisms, and the taxonomic diversity of microorganisms did not change even after additional disinfection with UV.Additional advanced wastewater treatments are necessary to completely remove AMRGs.The finding that AMRB and AMRGs that could impact the environment were effectively inactivated and/or removed during treatment could be a countermeasure to mitigate the environmental and human health impacts associated with the prevalence of AMR.The addition of the present on-site UV disinfection treatment system may be effective not only in regional places but also in developing regions and countries, due to the simplicity and convenience of preventing the potential spread of infectious diseases at the upstream stage in terms of drainage hygiene.Economic advantages are important considerations when developing practical applications.Further research is urgently required to prevent the spread of infectious diseases from wastewater.The overall results provide a better understanding of the current situation of AMR in hospital wastewater and provide insights for devising strategies to eliminate or mitigate the burden of AMRB flow into aquatic environments.

Figure 5 .
Figure 5. Taxonomic diversity of bacterial communities in the direct UV and UV-LED treated wastewater samples.((a) WWTP wastewater and (b) hospital effluent).

Figure 5 .
Figure 5. Taxonomic diversity of bacterial communities in the direct UV and UV-LED treated wastewater samples.((a) WWTP wastewater and (b) hospital effluent).

Table 2 .
The occurrence of AMRB and AMSB in the wastewater treatment plant (WWTP) influent, secondary effluent, effluent, model WWTP wastewater, and hospital effluent.

Table 3 .
Reaction rate constants for AMRB and AMSB during UV (254 nm) and UV-LED (280 nm) treatment of WWTP wastewater and hospital effluent.

Table 3 .
Reaction rate constants for AMRB and AMSB during UV (254 nm) and UV-LED (280 nm) treatment of WWTP wastewater and hospital effluent.

Table 4 .
Fluence required for 99% inactivation of AMRB and AMSB during UV (254 nm) and UV-LED (280 nm) treatment of WWTP wastewater and hospital effluent.